Although urothetium is a frequent site of tumor formation, relatively little is known about the molecular mechanisms of how urothelium is malignantly transformed by oncogenic events such as oncogene activation and tumor suppressor gene inactivation. During the last granting period, we studied bladder tumorigenesis by developing transgenic mouse models expressing several oncogenes under the direction of a urothelium-specific promoter. Urothelial expression of activated Ha-ras oncogene elicits urothelial hyperplasia followed by low-grade, superficial papillary tumors, whereas urothelial expression of SV40T oncogene induces high-grade, carcinoma in situ of the bladder, some of which progress into invasive and metastatic cancers. These results provide the first direct experimental evidence supporting the concept that bladder cancers arise and progress via distinctive phenotypic and genetic pathways. In the next granting period, we will examine the in vivo effects of inactivating several tumor suppressor genes on urothelial growth, differentiation and tumorigenesis, and will study the cooperative effects between oncogene-acfivation and tumor suppressor gene-inactivation on bladder tumor progression. Toward these goals, we will test the hypotheses that: (1) inactivation of p l6Ink4a tumor suppressor gene can synergize with Ha-ras activation to accelerate superficial papillary tumor formation; (2) inactivation of pi9Arf or p53 tumor suppressor gene can cooperate with Ha-ras activation to cause bladder tumor invasion; and (3) inactivation of both p53 and pRb tumor suppressor genes is required for the genesis of high-grade, invasive tumors of the bladder. We will employ a combination of transgenic, bi-transgenic, urothelium-specific gene knockout and double-knockout approaches to test these hypotheses. These studies will yield new insights regarding the molecular basis of bladder tumorigenesis, and the genetic causes of different bladder tumor progression pathways. The transgenic and knockout models that we will generate will serve as important in vivo platforms for evaluating novel diagnostic, preventive and therapeutic strategies for bladder cancers.